THE  HAEMOCYANIN  OF  LIMULUS  POLY- 
PHEMUS. 


BY 

C.  L.  ALSBERG  and  E.  D.  CLARK. 


(From  the  U.  S.  Bureau  of  Fisheries  Laboratory  at  Woods  Hole, 
Mass.,  and  the  Department  of  Biological  Chemistry  of  the 
Harvard  Medical  School.) 


From 

JOURNAL  OF  BIOLOGICAL  CHEMISTRY 
Vol.  VIII,  No.  1,  July,  1910 


i\  s>rvi 


i-m  „ 

^•\i 


REMOTE 

Reprinted  from  The  Journal  of  Biological  Chemistry,  vcfl.  ’Vifi, 


THE  HAEMOCYANIN  OF  LIMULUS  POLYPHEMUS. 

By  C.  L.  ALSBERG  and  E.  D.  CLARK. 

(From  the  U.  S.  Bureau  of  Fisheries  Laboratory  at  Woods  Hole,  Mass.,  and 
the  Department  of  Biological  Chemistry  of  the  Harvard  Medical 

School.) 

(Received  for  publication,  April  12,  1910.)  * 

The  question  whether  homologous  proteins  are  identical  in 
different  animals  is  one  of  considerable  general  interest.  It  has 
attracted  much  attention,  and  much  care  has  been  devoted  to  it 
by  Osborne1.  Abderhalden  and  Schittenbelm2  have  also  published 
upon  it.  The  former  compared  vegetable  proteins,  the  latter 
different  caseins.  Hsemocyanin  offers  a particularly  favorable  ma- 
terial for  such  a study,  since,  while  it  is  supposed  to  have  the  same 
functions  everywhere,  it  occurs  in  organisms  less  closely  related 
than  the  different  species  of  mammals  or  the  different  species  of 
legumes.  Henze3  has  made  a very  complete  study  of  the  hsemo- 
cyanin  of  the  octopus  ( Octopus  vulgaris) . He  has  succeeded  in  crys- 
talizing  it  by  the  original  method  of  Hofmeister  as  well  as  by  the 
Hopkins-Pinkus  modification,  and  he  has  determined  a good  many 
of  the  amino-acids  yielded  on  hydrolysis.  We  studied  the  hsemo- 
cyanin  of  Limulus  and  compared  our  results  with  those  of  Henze 
upon  Octopus.  Limulus  offers  exceptionally  good  material  for  a 
study  of  this  kind,  since  in  the  early  summer  it  is  very  abundant 
about  Woods  Hole,  and  a large  female  may  yield  as  much  as  400 
o'  cc.  of  blood. 

^ The  blood  was  obtained  by  making  an  incision  in  the  back  at 
the  joint  between  the  head  and  the  abdominal  piece.  The  yield 

K--]  1 Osborne  and  Clapp:  Amer.  Journ.  of  Physiol.,  xx,  p.  494;  Osborne  and 

Heyl:  Ibid.,  xxii,  p.  423. 

2 Zeitschr.  f.  physiol.  Chem.,  xxvii,  p.  458. 

3 M.  Henze:  Zur  Kenntniss  der  Haemocyanins,  Zeitschr.  f.  physiol.  Chem., 
xxxiii,  p.  370.  Zur  Kenntniss  der  Haemocyanins,  II.  Mitteilung,  Ibid., 
xliii  p.  290. 


i 


2 Haemocyanin  of  Limulus  Polyphemus 

of  blood  may  be  increased  by  doubling  up  and  straightening  out 
the  animal  at  this  joint,  like  opening  and  closing  a bellows,  thus 
squeezing  out  nearly  all  of  the  blood.  The  blood  was  allowed  to 
clot,  and  when  the  clot  had  contracted  the  serum  was  strained 
through  a cloth.  It  was  then  placed  in  the  ice-box  for  twelve  to 
twenty-four  hours  in  order  that  the  floccules,  which  we  have  des- 
cribed in  a previous  paper1  (and  which  Loeb2  before  us  has  shown 
simulate  a second  coagulation),  might  settle  out.  These  were 
filtered  off.  They  contain  a most  interesting  protein,  upon  which 
we  hope  to  report  before  long.  From  the  clear  serum  the  haemo- 
cyanin was  prepared  in  one  of  two  ways : 

(a)  It  was  dialyzed  until  all  the  haemocyanin  was  precipitated. 
The  precipitate  was  then  filtered  off ; dissolved  in  5 per  cent  sodium 
chloride  and  fractionated  with  ammonium  sulohate.  We  think 
this  is  not  a very  desirable  method,  for  the  haemocyanin  does  not 
always  precipitate  completely.  We  do  not  quite  understand  the 
conditions  for  the  complete  precipitation  of  haemocyanin  by  dialy- 
sis, but  we  are  under  the  impression  that  too  lengthy  dialysis 
causes  some  of  it  to  go  into  solution.  Moreover,  we  suspect,  as 
we  will  explain  later,  that  some  of  the  copper  may  be  lost  in  the 
process. 

(b)  The  serum  was  fractionated  with  ammonium  sulphate. 
We  believe  this  method  to  be  preferable.  As  the  serum  was  very 
alkaline  it  was  neutralized  carefully  with  very  weak  acetic  acid, 
not  stronger  than  .05  per  cent,  which  must  be  added  drop  by  drop, 
stirring  constantly.  If  the  acid  is  added  too  rapidly,  or  in  excess, 
the  haemocyanin  is  decomposed,  the  protein  part  precipitating  and 
more  or  less  copper  remaining  in  solution.  At  no  time  must  the 
serum  be  allowed  to  become  even  faintly  acid.  It  is  best  to  cease 
adding  acid  when  the  reaction  is  still  faintly  alkaline.  Under  these 
conditions  the  first  protein  precipitation  appeared  when  3.3 
parts  of  saturated  ammonium  sulphate  were  contained  in  a total 
volume  of  10  cc.  At  this  concentration  the  amount  precipitated 
was  slight.  It  was  bluish,  showing  that  it  contained  haemocyanin. 
At  a concentration  of  4.5  cc.  of  ammonium  sulphate  in  10  cc.  prac- 
tically all  the  haemocyanin  was  precipitated.  Traces  still  came 


1 This  Journal , v,  p.  323. 

2 Beitr.  z.  chem.  Physiol,  u.  Pathol .,  v,  p.  194. 


C.  L.  Alsberg  and  E.  D.  Clark 


3 


down  at  a concentration  of  4.7  cc.  in  10  cc.  Further  addition 
caused  no  precipitation  until  a concentration  of  5.5  cc.  in  10  cc. 
was  reached.  This  precipitate  was  not  very  voluminous.  It  was 
white,  and  therefore  not  hsemocyanin.  A last,  scanty  precipitate 
was  brought  down  by  complete  saturation  with  ammonium 
sulphate. 

All  in  all,  there  is  very  little  protein  in  the  blood  except  hsemocy- 
anin.  Indeed  we  believe,  though  we  have  not  yet  demonstrated  it 
as  a fact,  that  all  the  protein  present  except  the  hsemocyanin  is 
derived  from  the  disintegrating  cells  of  the  clot.  We  base  this  view 
on  the  observation  that  the  serum  collected  before  the  clot  has  begun 
to  contract  much  is  poorer  in  these  proteins  than  that  obtained 
after  full  contraction  of  the  clot.  We  did  not  pursue  this  line  of  in- 
vestigation further  because  it  ought  to  be  carried  out  on  animals 
before  spawning,  when  they  are  in  the  best  condition.  Our  animals 
were  used  later,  sometimes  after  having  been  kept  in  a floating  car 
in  the  harbor  several  weeks. 

We  therefore  precipitated  the  serum  with  80  cc.  of  saturated 
ammonium  sulphate  solution  for  each  100  cc.  of  serum.  The  crude 
hsemocyanin  thus  obtained  was  filtered  off,  dissolved  in  water,  and 
reprecipitated  with  ammonium  sulphate.  The  necessary  ammonium 
sulphate  was  added  slowly  until  a concentration  of  about  3.4  to  3.5 
in  10  cc.  was  attained.  The  precipitate  thus  obtained  was  filtered 
off  and  rejected.  Then  more  ammonium  sulphate  solution  was 
added  to  the  filtrate  until  a saturation  of  about  4.4  cc.  in  10  cc. 
was  reached.  The  precipitate  thus  formed  was  filtered  off  and  pre- 
served. In  this  way  the  material  precipitated  at  the  lower  and 
upper  limits  was  rejected,  the  presumption  being  that  contaminat- 
ing material  was  thereby  removed.  There  is  of  course  no  guaranty 
that  by  this  procedure  protein  with  the  same  precipitation  limits 
as  hsemocyanin  is  removed.  That  no  such  protein  occurs  in  the 
serum  we  are  not  prepared  to  say.  We  have  encountered  no  evi- 
dence of  its  existence.  The  hsemocyanin  precipitated  was  redis- 
solved and  reprecipitated  twice  more  in  this  fashion.  After  the 
first  precipitation  the  limits  of  salt  concentration  were  a little  higher 
than  they  were  in  the  original  serum,  possibly  because  the  serum 
itself  has  about  the  concentration  of  salts  found  in  sea-water. 
After  the  last  precipitation  the  hsemocyanin  was  redissolved  and 
precipitated  with  alcohol,  under  which  it  was  kept  for  some  time 


4 


Haemocyanin  of  Limulus  Polyphemus 


to  coagulate  it  thoroughly.  The  alcohol  was  then  decanted  off 
and  the  coagulum  washed  free  from  salt.  There  was  of  course 
the  danger  that  the  coagulum  might  retain  traces  of  the  sulphate. 
If,  however,  the  alcohol  were  added  slowly,  with  constant  stirring, 
a very  light  flocculent  precipitate  was  obtained  which  was  easily 
washed.  Moreover,  only  just  enough  alcohol  to  coagulate  should 
be  added,  so  as  to  keep  as  much  of  the  sulphate  in  solution  as  poss- 
ible. The  sulphate  may  of  course  be  dialzyed  away  before  adding 
the  alcohol,  and  thus  this  source  of  error  may  be  avoided.  How- 
ever, we  feared  losing  copper  (cf.  below)  more  than  adsorbing 
salt.  The  material  thus  obtained  was  analyzed,  after  having  been 
powdered  and  dried  to  constant  weight  in  vacuo  over  sulphuric 
acid  at  a temperature  of  70°.  It  gave  the  following  figures: 

0.2174  gm.  substance  Preparation  I yielded  0.1405  gm.  H20  and  0.3891  gm. 
C02: 

H = 7.18  per  cent. 

C = 48.80  per  cent. 

0.2094  gm.  substance  Preparation  II  yielded  0.1324  gm.  H20  and  0.3769 
gm.  C02: 

H = 7.02  per  cent 

C = 49.09  per  cent. 

0.1950  gm.  substance  Preparation  I yielded  27.30  cc.  N at  18°  and  766  mm. : 

N = 16.30  per  cent 

0.2221  gm.  substance  Preparation  II  yielded  31.02  cc.  N at  19°  and  760.5 


N = 16.06  per  cent 

Sulphur  was  determined  according  to  Folin.  0.4086  gm.  substance  Prepar- 
ation I yielded  0.0465  gm.  BaS04: 

S = 1.56  per  cent 

Copper  was  determined  by  incinerating  in  porcelain,  and  exhaust' 
ing  the  ash  with  nitric  acid.  A trace  of  material,  probably  silica, 
did  not  dissolve.  The  presence  of  silica  in  the  clot  has  already 
been  reported  by  us1.  The  acid  solution  was  evaporated  to  dryness 
with  a little  H2S04.  The  solution  taken  up  in  a little  very  dilute 
H2S04  was  filtered  and  the  copper  determined  electrolytically. 

0.5115  gm.  substance  Preparation  I yielded  0.0015  gm.Cu: 

Cu  = 0.29  per  cent. 

For  the  check  analysis  a little  more  material  was  taken  and  a 
different  method  used.  The  material  in  this  case  was  dried  to 


1Loc.  cit. 


C.  L.  Alsberg  and  E.  D.  Clark 


5 


constant  weight  in  the  oven  at  105°.  The  ash  was  exhausted  with 
nitric  acid,  the  nitric  replaced  with  hydrochloric  acid,  the  copper 
precipitated  as  the  sulphide,  the  latter  collected  on  filter  paper, 
washed,  ignited,  converted  into  the  nitrate,  and  the  latter  into  the 
oxide  by  ignition. 

0.9960  gm.  substance  Preparation  II  yielded  0. 0034  gm.  CuO : 

Cu  = 0.0027  gm.  = 0.27  per  cent 

The  hsemocyanin  obtained  by  Henze  from  the  octopus  had  a 
considerably  different  composition.  The  following  are  his  results 
and  ours  side  by  side: 


OCTOPUS. 

LIMULUS. 

(Average) 

(Average) 

per  cent. 

per  cent. 

c.. 

53.66 

48.94 

H.. 

7.33 

7.10 

N.. 

16.09 

16.18 

S... 

0.86 

1.56 

Cu. 

0.38 

0.28 

O.. 

21.68 

25.94 

It  would  seem  from  this  comparison  that  the  two  substances  are 
different.  The  greatest  difference  is  in  the  sulphur  content  which 
is  too  great  to  be  due  to  differences  in  the  methods  of  determination 
used.  The  difference  in  the  copper  content  is  also  condsiderable  if 
we  assume  that  our  values  are  correct.  We  realize  fully  that  to 
establish  them  we  need  more  determinations  with  larger  quantities 
of  material.  Perhaps  it  may  be  found  that  animals  more  sluggish 
than  the  octopus  possess  haemocyanins  of  lower  copper  content,  and 
therefore  less  active  oxygen  carriers.  Certainly  their  blood  as 
a whole  usually  contains  less  copper.1  The  difference  in  the  carbon 
content  of  our  preparation  as  compared  with  that  of  Henze  is 
also  great,  and  argues  for  the  individuality  of  this  substance. 

It  is,  of  course,  possible  that  the  difference  in  our  figures  is  due  to 
the  fact  that  we  worked  with  an  impure  substance.  We  do  not 
regard  this  as  probable,  although  we  must  admit  that  we  have  not 
the  same  guaranty  of  purity  that  Henze  had,  for  we  were 
quite  unable  to  crystallize  this  substance  as  he  did  his.  The 
Hofmeister  method  failed  us,  while  in  its  Hopkins-Pinkus  modifi- 


1 Cf.  v.  Fiirth:  V ergleichende  chemische  Physiologie  der  niederen  Tiere. 


6 


Haemocyanin  of  Limulus  Polyphemus 


cation  it  could  not  be  used  at  all  because  of  the  great  sensitiveness 
of  this  substance  to  acids.  If  to  the  solution  we  added  a few  drops 
of  acetic  acid,  as  Henze  did,  we  got  a heavy,  curdy  precipitate  which 
could  not  be  brought  back  into  solution  except  by  the  use  of  alkali. 
We  tried  to  obtain  this  substance  in  crystalline  form,  both  from  the 
fresh  serum  as  well  as  from  purified  haemocyanin.  We  are  well 
aware  our  negative  results  do  not  prove  that  the  substance  is 
uncrystal liz able.  The  conditions  are  not  as  favorable  for  its  crystal- 
lization in  Limulus  as  they  seem  to  be  in  Octopus.  It  is  so  difficult  to 
prevent  larger  quantities  of  the  blood  from  clotting  that  we  were 
compelled  to  work  with  the  serum  instead  of  centrifugated  blood. 
The  process  of  clotting  as  shown  by  Loeb1  consists  of  an  agglutina- 
tion of  cells,  accompanied  by  their  disintegration.  This,  as  already 
indicated,  we  believe  to  be  accompanied  by  the  passage  of  material 
from  the  cells  into  the  serum,  which  might  well  interfere  with 
crystallization.  Furthermore,  Henze2  states  that  crystallization 
goes  on  well  only  with  fresh  blood  from  healthy  animals.  While 
we  used  fresh  blood,  we  had  to  lose  some  time  in  getting  rid  of  the 
clot.  Moreover,  our  animals,  while  apparently  in  good  condition, 
had  been  collected  and  kept  in  a floating  car  in  the  harbor  several 
weeks  before  they  were  used.  Possibly  with  animals  taken  before 
spawning  and  used  immediately  the  result  might  have  been  differ- 
ent. While  these  considerations  may  account  for  our  inability  to 
obtain  crystals,  we  are  inclined  to  believe  that  the  real  reason  is 
that  Limulus  haemocyanin  is  a globulin. 

So  much  for  our  quantitative  studies,  which  all  point  to  the  indi- 
viduality of  Octopus  and  Limulus  haemocyanin.  Our  qualitative 
studies  offer  more  evidence  for  the  same  conclusion. 

Henze  states  that  his  haemocyanin  could  not  be  precipitated  by 
dialysis,  and  questions  the  results  of  Halliburton,  who  reported 
that  it  could  be  so  precipitated.  Henze  does  not  seem  to  have 
taken  into  consideration  that  there  might  be  more  than  one  kind 
of  haemocyanin  in  nature,  and  that  both  he  and  Halliburton  might 
be  right,  each  for  the  haemocyanin  with  which  he  worked.  As  a 
matter  of  fact,  Limulus  haemocyanin  may  be  completely  precipitated 
from  serum  by  dialysis.  Solutions  of  pure  haemocyanin  are  not  as 

1 Loc.  cit. 

2 Loc.  cit.,  p.  374. 


C.  L.  Alsberg  and  E.  D.  Clark 


7 


easily  completely  precipitated,  and  occasionally  behave  in  an  erratic 
way  that  we  have  not  yet  explained  to  our  own  satisfaction, 
though  we  think  it  was  probably  resolution  from  too  long  disalysis. 
Always,  however,  the  greater  part  was  precipitated.  Our  haemo- 
cyanin  behaved  like  a globulin,  Henze’s  like  an  albumen.  Our  haemo- 
cyanin  could  be  completely  precipitated  by  magnesium  sulphate, 
Henze’s  could  not  be.  4.7  cc.  saturation  with  ammonium  sulphate 
completely  precipitates  it.  Henze’s  is  only  precipitated  by  com- 
plete saturation.  In  this  respect,  too,  Limulus  haemocyanin  be- 
haves like  a globulin,  Octopus  haemocyanin  like  an  albumen.  By 
carbon  dioxide  our  haemocyanin  was  only  incompletely  precipitated, 
agreeing  in  this  respect  with  Henze’s.  It  gave  the  protein  precipi- 
tation reactions,  the  xanthoproteic,  the  biuret,  and  Mi  lion’s  re- 
action. It  was  precipitated  by  the  salts  of  the  heavy  metals. 

We  are  not  certain  how  far  the  behavior  of  Limulus  haemocyanin 
toward  acids  differs  from  that  of  Octopus  haemocyanin,  for  we  do 
not  quite  understand  (no  doubt  our  own  fault)  how  the  statements 
of  Henze,  on  pp.  377  and  380  of  his  first  paper,  are  to  be  reconciled 
with  one  another.  We  get  the  general  impression  from  reading  his 
papers  that  our  substance  was  more  sensitive  to  acid  than  his. 
Very  slight  traces  of  dilute  acid,  including  acetic,  precipitated  it 
as  white  floccules  poor  in  copper.  Further  addition  of  acid  did 
not  redissolve  it.  The  copper  is  in  exceedingly  loose  combination.1 
It  may  even  be  completely  removed  by  dialysis  if  the  water  used 
be  kept  very  faintly  acid.  The  dialysis  must  be  very  protracted, 
extending  over  a number  of  days.  If  enough  salt  be  present,  the 
protein  will  not  be  precipitated.  A cold  storage  room,  kept  just 
above  zero,  enabled  us  to  carry  out  this  experiment  without  bac- 
terial action. 

The  possibility  of  losing  copper  in  this  way  kept  us  from  employ- 
ing dialysis  as  a step  in  preparation.  We  have  convinced  our- 
selves that  lengthy  dialysis  sometimes,  not  always,  results  in  a 
loss  of  copper  even  when,  apparently,  the  solution  has  remained 

1 One  of  us  has  already  called  attention  to  the  ease  with  which  copper  is  re- 
moved from  Limulus  haemocyanin  (C.  L.  Alsberg : Beitrage  zur  Kenntniss 
der  Guajakreaktion.  Arch.f.  exp.  Path.  u.  Pharmak.,  Supplementband, 
“Schmiedeberg  Festschrift,’’  1908,  p.  39);  and  he  has  also  suggested  (ibid., 
pp.  41-42)  that  different  haemocyanins  may  differ  in  their  susceptibility  to 
acid. 


8 


Haemocyanin  of  Limulus  Polyphemus 


neutral.  We  spoiled  considerable  material  before  we  realized 
this  possibility.  One  preparation  was  dialyzed  five  days  in  the 
cold  storage  room  at  a temperature  but  slightly  above  zero.  It 
gave  us  the  following  figures  for  copper: 


2.7999  gm.  substance  Preparation  III  yielded  0.0040  gm.  Cu  (electrolytic 
method) : 


Cu  = 0.143  per  cent 


To  make  certain  that  the  figures  were  correct,  a larger  quantity 
of  material  was  used,  and  the  sulphide  method  employed: 

II 

16.434  gm.  substance  Preparation  III  yielded  0.0291  gm.  CuO: 

Cu  = 0.138  per  cent 

Like  Henze,  we  were  unable  to  obtain  any  copper  compound 
analogous  to  haematin.  We  regard  our  substance  as  a copper 
globulin  compound,  while  he  regards  his  as  a copper  albuminate. 
We  believe  they  are  probably  two  different  proteins,  though  we  are 
well  aware  that  this  can  be  settled  finally  only  by  a quantitative 
hydrolysis  for  which  we  lack  at  present  sufficient  material. 


SUMMARY. 

The  haemocyanin  of  Limulus  differs  from  that  of  Octopus  in 
percentage  composition,  in  its  precipitability  by  dialysis,  by  full 
saturation  with  magnesium  sulphate  and  by  half  saturation  with 
ammonium  sulphate,  in  not  having  been  crystallized,  and  perhaps  in 
being  more  sensitive  to  acid.  There  are  therefore  different  haemo- 
cyanins,  and  perhaps  this  fact  accounts  for  the  discrepancies  in 
the  literature  concerning  the  properties  of  this  substance. 


WILLIAMS 


PRESS  OF 

& WILKINS  COMPANY 
BALTIMOR  E 


12  10606798; 


